NONLINEAR DECOMPOSITION OF HEME UV-VIS SPECTRA

我们建议将一系列分析工具扩展和组合到一般情况下 在典型样品紫外-可见光谱解释和分析中的应用 化学和生化系统。数字化的广泛使用，快速- 研究人员扫描分光光度计，研究范围广泛 问题，已经产生了对开发和提供 这些技巧。这一发展将通过研究实现 一系列逐渐复杂的血红素(铁卟啉)配体 均衡。第一个要研究的系统是滴定 均四苯基卟啉铁(II)，铁(II)TPP，与1，2-二甲基咪唑， 1，2-DMI。因为这个碱基只结合一次-在中等浓度-它的 滴定由两部分组成：Fe(II)TPP和Fe(II)TPP(1，2-DMI)。这个 两个相应的纯UV-Vis光谱很容易获得，因此是标准的 将使用方法学来重现结合常数。《光谱》 该滴定曲线将进一步处理如下：1) 因子分析分解(FA)，以及2)对一系列 高斯/洛伦兹函数(GLF)。使用这些分析背后的目标是 是为了确定每个人都可以复制的准确性；a)装订 常数，以及b)单独的纯光谱。除 这两种分析的统计方法，这两种 技术使用非线性最小二乘曲线拟合程序。这个 MarQuardt算法是一种非常有效的方法，但可能会失败 如果初始猜测关闭，则为全局最小值。改进的方法 对参数空间的搜索进行了探索。 在关于两个组件系统的工作之后，FA和GLF技术将 推广到由三、四、六、九组成的系统 组件。这些系统将由不同的组合组成 铁卟啉Fe(II)TPP和中位体(α，α-O- 吡咯烷胺卟啉铁(II)，铁(II)(TpivPP)，“栅栏”血红素，以及 碱基1-甲基咪唑，1，2-二甲基咪唑，1，5- 二环己基咪唑、吡啶和4-苯基吡啶。因为有几个 纯光谱和结合常数是已知的，或者它们的估计是 ，建议分析的发展和准确性将是 在每一步都进行了测试和严格的评估。要使用的配体是 所有的商业上都可以买到，除了一种外， “栅栏”卟啉，其合成和提纯是一系列 2高产率步骤：1)中-α， 从商品中分离得到的α-邻氨基苯基卟啉 通过柱层析可获得阿托品的混合物。2)插入 在回流过程中将FeBr加入到该卟啉中，将铁添加到该卟啉中 THF/CHCl3. 将为大型机和微型计算机开发软件。这个 暂定的时间表是建立实验室，并对两个和三个 第一年的组件系统。扩展到更复杂的 系统将占用第二年和第三年的大部分时间。跟随 几年后，该软件将应用于不同的系统 生化兴趣。

We propose to extend and combine a series of analytical tools for general use in the interpretation and analysis of UV-visible spectra of typical chemical and biochemical systems. The widespread use of digitizing, fast- scanning spectrophotometers by investigators studying a wide range of problems, has created the need for the development and availability of these techniques. This development will be brought about through the study of a series of progressively more complicated heme (iron porphyrin)-ligand equilibria. The first system to be studied is the titration of mesotetraphenylporphyrinatoiron(II), Fe(II)TPP, with 1,2-dimethylimidazole, 1,2-DMI. Since this base only binds once-at moderate concentrations-its titration consist of two components; Fe(II)TPP and Fe(II)TPP(1,2-DMI). The two corresponding pure UV-vis spectra are easily obtainable and so standard methodologies will be used to reproduce the binding constant. The spectral curves of this titration will then be further treated as follows: 1) Factor Analytical decomposition (FA), and 2) Fitting to a series of Gaussian/Lorentzian functions (GLF). The goal behind using these analysis is to determine the accuracy with which each can reproduce; a) the binding constant, and b) the individual pure spectra. In addition to the statistical methods of each of these two analysis, each of these two techniques uses nonlinear least-squares curve fitting procedures. The Marquardt algorithm is a very efficient method of doing this, but may miss global minima if the initial guesses are off. Methods for improved searching of parameter space will be explored. Following work on the two component system the FA and GLF techniques will be extended to systems composed of three, four, six and finally nine components. These systems will be composed of various combination of the iron porphyrins Fe(II)TPP and meso-tera(alpha, alpha, alpha, alpha-o- pivalamidoporphyrinatoiron(II), iron(II)(TpivPP), "picket fence" heme, and the bases 1-Methylimidazole, 1,2-dimethylimidazole, 1,5- dicyclohexylimidazole, pyridine and 4-phenylpyridine. Since several of the pure spectra and binding constants are known, or estimates of them are available, the development and accuracy of the proposed analyses will be tested, and critically evaluated, at each step. The ligands to be used are all commercially available as are the porphyrins with the exception of one, "picket fence" porphyrin, whose synthesis and purification is a series of 2 high yield steps: 1) condensation of meso-tera-alpha, alpha, alpha, alpha-o-aminopnenylporphyrin which is isolated form the commercially available mixture of atropisomers via column chromatography. 2) Insertion of iron to this porphyrin by adding FeBr to this porphyrin in refluxing THF/CHCl3. Software will be developed for both mainframe and microcomputers. The tentative timetable is to set up the lab and characterize the two and three component systems in the first year. Extensions to the more complicated systems will take up the second year and most of the third. Following years will see applications of the software to different systems of biochemical interest.